Evidence of a causal and modifiable relationship between kidney function and circulating trimethylamine N-oxide.

The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied "explainable" machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.

Metabolomic and microbiome profiling reveals personalized risk factors for coronary artery disease.

Complex diseases, such as coronary artery disease (CAD), are often multifactorial, caused by multiple underlying pathological mechanisms. Here, to study the multifactorial nature of CAD, we performed comprehensive clinical and multi-omic profiling, including serum metabolomics and gut microbiome data, for 199 patients with acute coronary syndrome (ACS) recruited from two major Israeli hospitals, and validated these results in a geographically distinct cohort. ACS patients had distinct serum metabolome and gut microbial signatures as compared with control individuals, and were depleted in a previously unknown bacterial species of the Clostridiaceae family. This bacterial species was associated with levels of multiple circulating metabolites in control individuals, several of which have previously been linked to an increased risk of CAD. Metabolic deviations in ACS patients were found to be person specific with respect to their potential genetic or environmental origin, and to correlate with clinical parameters and cardiovascular outcomes. Moreover, metabolic aberrations in ACS patients linked to microbiome and diet were also observed to a lesser extent in control individuals with metabolic impairment, suggesting the involvement of these aberrations in earlier dysmetabolic phases preceding clinically overt CAD. Finally, a metabolomics-based model of body mass index (BMI) trained on the non-ACS cohort predicted higher-than-actual BMI when applied to ACS patients, and the excess BMI predictions independently correlated with both diabetes mellitus (DM) and CAD severity, as defined by the number of vessels involved. These results highlight the utility of the serum metabolome in understanding the basis of risk-factor heterogeneity in CAD.

Microbiome and metabolome features of the cardiometabolic disease spectrum.

Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.

UCP1 expression-associated gene signatures of human epicardial adipose tissue.

Multiple reports of uncoupling protein 1 (UCP1) expression have established its presence in human epicardial adipose tissue (eAT). Its functional relevance to eAT, however, remains largely unknown. In a recent study, we reported that adrenergic stimulation of eAT was associated with downregulation of secreted proteins involved in oxidative stress-related and immune-related pathways. Here, we explored the UCP1-associated features of human eAT using next-generation deep sequencing. Paired biopsies of eAT, mediastinal adipose tissue (mAT), and subcutaneous adipose tissue (sAT) obtained from cardiac surgery patients, with specific criteria of high and low expression of UCP1 in eAT, were subjected to RNA sequencing. Although eAT exhibited a depot-specific upregulation in the immune-related pathways relative to mAT and sAT, high UCP1 expression in eAT was specifically associated with differential gene expression that functionally corresponded with downregulation in the production of reactive oxygen species and immune responses, including T cell homeostasis. Our data indicate that UCP1 and adaptive immunity share a reciprocal relationship at the whole-transcriptome level, thereby supporting a plausible role for UCP1 in maintaining tissue homeostasis in human eAT.

Brown and beige adipose tissues: phenotype and metabolic potential in mice and men.

With the recent rediscovery of brown fat in adult humans, our outlook on adipose tissue biology has undergone a paradigm shift. While we attempt to identify, recruit, and activate classic brown fat stores in humans, identification of beige fat has also raised the possibility of browning our white fat stores. Whether such transformation of human white fat depots can be achieved to enhance the whole body oxidative potential remains to be seen. Evidence to date, however, largely points toward a major oxidative role only for classic brown fat depots, at least in rodents. White fat stores seem to provide the main fuel for sustaining thermogenesis via lipolysis. Interestingly, molecular markers consistent with both classic brown and beige fat identity can be observed in human supraclavicular depot, thereby complicating the discussion on beige fat in humans. Here, we review the recent advances made in our understanding of brown and beige fat in humans and mice. We further provide an overview of their plausible physiological relevance to whole body energy metabolism.

Functional characterization of the Ucp1-associated oxidative phenotype of human epicardial adipose tissue.

Brown fat presence and metabolic activity has been associated with lower body mass index, higher insulin sensitivity and better cardiometabolic profile in humans. We, and others, have previously reported the presence of Ucp1, a marker of brown adipocytes, in human epicardial adipose tissue (eAT). Characterization of the metabolic activity and associated physiological relevance of Ucp1 within eAT, however, is still awaited. Here, we validate the presence of Ucp1 within human eAT and its 'beige' nature. Using in-vitro analytical approaches, we further characterize its thermogenic potential and demonstrate that human eAT is capable of undergoing enhanced uncoupling respiration upon stimulation. Direct biopsy gene expression analysis reveals a negative association between thermogenic markers and oxidative stress-related genes in this depot. Consistently, isoproterenol (Iso) stimulation of eAT leads to a downregulation of secreted proteins included in the GO terms 'cell redox homeostasis' and 'protein folding'. In addition, cardiac endothelial cells exhibit a downregulation in the expression of adhesion markers upon treatment with Iso-stimulated eAT derived conditioned media. Overall, these observations suggest that Ucp1- associated metabolic activity plays a significant role in local tissue homeostasis within eAT and can plausibly alter its communication with neighboring cells of the cardiovascular system.

Metabolic activity of brown, "beige," and white adipose tissues in response to chronic adrenergic stimulation in male mice.

Classical brown adipocytes such as those found in interscapular brown adipose tissue (iBAT) represent energy-burning cells, which have been postulated to play a pivotal role in energy metabolism. Brown adipocytes can also be found in white adipose tissue (WAT) depots [e.g., inguinal WAT (iWAT)] following adrenergic stimulation, and they have been referred to as "beige" adipocytes. Whether the presence of these adipocytes, which gives iWAT a beige appearance, can confer a white depot with some thermogenic activity remains to be seen. In consequence, we designed the present study to investigate the metabolic activity of iBAT, iWAT, and epididymal white depots in mice. Mice were either 1) kept at thermoneutrality (30°C), 2) kept at 30°C and treated daily for 14 days with an adrenergic agonist [CL-316,243 (CL)], or 3) housed at 10°C for 14 days. Metabolic activity was assessed using positron emission tomography imaging with fluoro-[(18)F]deoxyglucose (glucose uptake), fluoro-[(18)F]thiaheptadecanoic acid (fatty acid uptake), and [(11)C]acetate (oxidative activity). In each group, substrate uptakes and oxidative activity were measured in anesthetized mice in response to acute CL. Our results revealed iBAT as a major site of metabolic activity, which exhibited enhanced glucose and nonesterified fatty acid uptakes and oxidative activity in response to chronic cold and CL. On the other hand, beige adipose tissue failed to exhibit appreciable increase in oxidative activity in response to chronic cold and CL. Altogether, our results suggest that the contribution of beige fat to acute-CL-induced metabolic activity is low compared with that of iBAT, even after sustained adrenergic stimulation.

Understanding the brown adipocyte as a contributor to energy homeostasis.

Brown adipocytes are specialized cells capable of undergoing thermogenesis, a phenomenon regulated by the sympathetic nervous system, due to the presence of uncoupling protein 1 (UCP1). The recent demonstrations of their presence in adult humans, and the discovery that brown adipocytes can be derived from distinct precursors and express specific genes depending on their anatomic location, have sparked intense interest in enhancing the current understanding of their biology and relevance to human energy homeostasis. We provide an overview of the latest advances related to the developmental origins of brown adipocytes, discuss their regulation and function in both rodents and humans, and offer a critical perspective on the relevance of brown adipocyte-mediated thermogenesis in human physiology.

Brown fat like gene expression in the epicardial fat depot correlates with circulating HDL-cholesterol and triglycerides in patients with coronary artery disease.

BACKGROUND: Recent evidence indicates that epicardial adipose tissue (EAT) expresses uncoupling protein-1 (UCP1), a marker of brown adipocytes. However, the putative effects of the presence of brown adipocytes in EAT remain unknown. METHODS: The mRNA expression of genes related to brown adipocyte-mediated thermogenesis was measured in the fat samples collected from the epicardial-, mediastinal- and subcutaneous-depots of patients undergoing coronary artery bypass grafting. Both univariate and multivariate analyses were then utilized to determine any association between gene expression and the anthropometrics and fasting blood chemistries of these patients. RESULTS: EAT exhibited significantly higher expression of UCP1 and cytochrome c oxidase subunit-IV (COX-IV) compared to mediastinal- and subcutaneous-fat depots (P ≤ 0.05). EAT expression of UCP1 (r=0.50), COX-IV (r=0.37) and lipoprotein lipase (LPL) (r=0.58) positively associated with circulating levels of HDL-cholesterol (P ≤ 0.05). In addition, EAT expression of LPL, acyl coA dehydrogenase-short, -medium and -long chain genes associated negatively with circulating TG levels (P ≤ 0.05). CONCLUSIONS: Abundance of UCP-1 in the EAT relative to other fat depots confirms the presence of brown adipocytes in human EAT. Furthermore, the correlations among the EAT expression of thermogenesis-related genes with the circulating HDL and TG levels indicate that presence of active brown adipocytes shares a functional association with the circulating plasma lipids in humans.

Control and physiological determinants of sympathetically mediated brown adipose tissue thermogenesis.

Brown adipose tissue (BAT) represents a remarkable heat-producing tissue. The thermogenic potential of BAT is conferred by uncoupling protein 1, a protein found uniquely in brown adipocytes. BAT activity and capacity is controlled by the sympathetic nervous system (SNS), which densely innervates brown fat depots. SNS-mediated BAT thermogenesis is essentially governed by hypothalamic and brainstem neurons. BAT activity is also modulated by brain energy balance pathways including the very significant brain melanocortin system, suggesting a genuine involvement of SNS-mediated BAT thermogenesis in energy homeostasis. The use of positron emission tomography/computed tomography scanning has revealed the presence of well-defined BAT depots in the cervical, clavicular, and paraspinal areas in adult humans. The prevalence of these depots is higher in subjects exposed to low temperature and is also higher in women compared to men. Moreover, the prevalence of BAT decreases with age and body fat mass, suggesting that BAT could be involved in energy balance regulation and obesity in humans. This short review summarizes recent progress made in our understanding of the control of SNS-mediated BAT thermogenesis and of the determinants of BAT prevalence or detection in humans.

Validation of reference genes for the relative quantification of gene expression in human epicardial adipose tissue.

BACKGROUND: Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. METHODOLOGY/PRINCIPAL FINDINGS: Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CONCLUSIONS/SIGNIFICANCE: CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue.

Maternal dietary fat intake during gestation and lactation alters tissue fatty acid composition in the adult offspring of C57Bl/6 mice.

We investigated the effects of maternal dietary fat intake during gestation and lactation on the tissue fatty acid composition of the adult offspring. Female C57Bl/6 mice were fed high fat diets enriched with lard or safflower oil or chow during mating, gestation and lactation. The offspring obtained from each group of mothers were continued on diets rich in lard, safflower oil or chow post-weaning until 11 weeks of age. Livers and hearts were collected for fatty acid analysis. A maternal diet rich in safflower oil was associated with enrichment of hepatic tissue with n-3 polyunsaturated fatty acids in the offspring fed chow post-weaning compared to the offspring fed chow throughout. However, a continuous exposure to a safflower oil- as well as lard-rich diet during the pre- and post-weaning time periods was associated with reduced content of docosahexaenoic acid in both liver and heart tissues compared to the offspring fed chow throughout. In conclusion, this study demonstrated lasting effects of maternal dietary fat intake, as well as an interaction between pre- and post-weaning diets, on the tissue fatty composition in adult offspring.

Flax oil-mediated activation of PPAR-γ correlates with reduction of hepatic lipid accumulation in obese spontaneously hypertensive/NDmcr-cp rats, a model of the metabolic syndrome.

Flax oil feeding has been proposed to have beneficial effects on the outcome of the metabolic syndrome due to the high n-3 fatty acid content of flax oil; however, the mechanisms of its action remain largely unknown. We investigated the effects of flax oil feeding on hyperlipidaemia, hyperglycaemia, hepatic steatosis and oxidative stress in the spontaneously hypertensive (SHR)/NDmcr-cp rats, a genetic model of the metabolic syndrome. Hepatic gene expression of PPAR-α, PPAR-γ and sterol-regulatory element-binding protein-1c was also assessed in order to investigate the possible underlying mechanisms. Obese and lean SHR/NDmcr-cp rats were fed high-fat diets enriched with either lard or flax oil for a period of 4 weeks. Obese rats exhibited higher body weight, liver weight and mesenteric fat-, epididymal fat- and renal fat-pad weights, and also TAG and cholesterol concentrations in serum and VLDL, LDL and HDL fractions, when compared with the lean rats (P < 0·001), irrespective of the diets. Concentrations of fasting serum insulin and urinary thiobarbituric acid reactive substances were lower in flax oil-fed obese (FO) rats compared with the lard-fed obese (LO) rats (P < 0·01). Flax oil feeding also revealed a significant reduction in hepatic TAG and cholesterol concentrations in obese rats compared with the LO rats (P < 0·05). In addition, FO rats exhibited significantly higher hepatic mRNA expression of PPAR-γ, which negatively correlated (r - 0·98, P < 0·05) with their hepatic lipid levels. These findings suggest that flax oil feeding may activate PPAR-γ-dependent pathways to alter the hepatic lipid metabolism and to increase insulin sensitivity in the obese SHR/NDmcr-cp rats.

An interaction of the pre- and post-weaning diets rich in omega-6 polyunsaturated fats alters plasma lipids, hepatic gene expression and aortic vascular reactivity in adult C57BL/6 mice.

AIM: To investigate the effects of diets rich in n-6 polyunsaturated fats (PUFA) fed during pre- and post-weaning time periods on the lipid metabolism and vascular reactivity in adult C57Bl/6 mice, in order to assess the impact of maternal nutrition and its interaction with the offspring diet on the metabolism of adult offspring. METHODS: Female C57Bl/6 mice were fed a high-fat diet enriched with n-6 PUFA (P) or control diet (C) for 2-weeks before, during mating, gestation and lactation, while their pups received either P or C for 8-weeks post-weaning. RESULTS: A significant interaction between the maternal and post-weaning diets was observed for the offspring body weight, food-, caloric-intake, plasma lipids, hepatic mRNA expression of lecithin cholesterol acyltransferase, aortic contractile and relaxation responses (P < 0.05). CONCLUSION: The overall metabolic and physiological outcome in the offspring is dependent upon the interaction between the pre- and post-weaning dietary environments.

Developmental programming of lipid metabolism and aortic vascular function in C57BL/6 mice: a novel study suggesting an involvement of LDL-receptor.

We have previously shown that a maternal high-fat diet, rich in saturated fatty acids (SFA), alters the lipid metabolism of their adult offspring. The present study was designed to investigate 1) whether alterations in hepatic LDL-receptor (LDL-r) expression may serve as a potential mechanism of developmental programming behind the altered lipid metabolism of the offspring, 2) whether altered lipid metabolism leads to aortic vascular dysfunction in the offspring, 3) whether deleterious effects of SFA exposure preweaning are influenced by postweaning diet, and 4) whether gender-specific programming effects are observed. Female C57Bl/6 mice were fed a high-SFA diet or regular chow during gestation and lactation while their pups, both male and female, received either SFA or a chow diet after weaning. Male offspring obtained from mothers fed an SFA diet and those who continued on chow postweaning had higher plasma triglycerides and total cholesterol, whereas female offspring had higher plasma total and LDL cholesterol levels, lower hepatic LDL-r mRNA expression, and reduced aortic contractile responses compared with the offspring that were fed chow throughout the study. A comparison of the postweaning diet revealed significantly lower hepatic LDL-r expression along with significantly higher plasma LDL-cholesterol concentration in the female offspring that were obtained from mothers fed an SFA diet and who continued on an SFA diet postweaning, compared with the female offspring that were obtained from mothers fed an SFA diet but who continued on chow postweaning. In conclusion, we report a novel observation of hepatic LDL-r-mediated programming of altered lipid metabolism, along with aortic vascular dysfunction, in the female offspring of mothers fed a high-SFA diet. Male offspring only exhibited dyslipidemia, suggesting gender-mediated programming. This study further highlighted the role of postweaning diets in overriding the effects of maternal programming.

Maternal diet rich in saturated fats has deleterious effects on plasma lipids of mice.

BACKGROUND AND OBJECTIVES: High dietary fat intake has been reported to cause an alteration in lipid metabolism that is associated with an increased risk of cardiovascular disease. In the present study, an animal model was used to evaluate the effects of feeding diets rich in different fatty acids to mothers during pregnancy and lactation, and the effects of the maternal diet on parameters of lipid metabolism in adult offspring. The interaction between the offspring's own diet and the programming due to the maternal diet was also evaluated. METHODS: Female C57BL/6 mice were fed a high-fat diet (20% fat [weight to weight]) rich in either saturated fatty acids (SFA) or polyunsaturated fatty acids (PUFA) for two weeks before mating, during pregnancy and until weaning. The offspring were divided into two groups; each group was fed a high-fat diet enriched in either SFA or PUFA for eight weeks after weaning. The groups were designated as SFA/SFA (diet of the mother/diet of the offspring), SFA/PUFA, PUFA/PUFA and PUFA/SFA. Blood and tissues were collected at the end of the eight-week feeding period after an overnight fast. RESULTS: The plasma total cholesterol and low density lipoprotein cholesterol concentrations were significantly higher in the SFA/SFA group than in all other groups, whereas the PUFA/PUFA group had the lowest total cholesterol and low density lipoprotein cholesterol concentrations. Plasma high density lipoprotein cholesterol concentrations were significantly higher in the PUFA/SFA group than in the PUFA/PUFA and SFA/PUFA groups, whereas plasma triglyceride concentrations were not different among the groups. CONCLUSIONS: The data suggest that high maternal dietary fat intake during pregnancy affects lipid metabolism in the adult offspring. However, it appears that the offspring's own diet is also important in maintaining the regulation of lipid metabolism.